Protocadherin PAPC is expressed in the CNC and can compensate for the loss of PCNS

Protocadherins represent the biggest subgroup within the cadherin superfamily of transmembrane glycoproteins. In contrast to classical type I cadherins, protocadherins in general exhibit only moderate adhesive activity. During embryogenesis, they are involved in cell signaling and regulate diverse morphogenetic processes, including morphogenetic movements during gastrulation and neural crest migration. The two protocadherins paraxial protocadherin (PAPC) and axial protocadherin (AXPC) are indispensable for proper gastrulation movements in Xenopus and zebrafish. The closest relative PCNS instead, is required for neural crest and somite formation. Here, we show that cranial neural crest (CNC) cells in addition to PCNS express PAPC, but not AXPC. Overexpression of PAPC resulted in comparable migration defects as knockdown of PCNS. Moreover, reconstitution experiments revealed that PAPC is able to replace PCNS in CNC cells, indicating that both protocadherins can regulate CNC migration. genesis 52:120–126. © 2013 Wiley Periodicals, Inc.     

Chondroitin 4‐O‐sulfotransferases are required for cell adhesion and morphogenesis in the Ciona intestinalis embryo

Chondroitin sulfate (CS) is a carbohydrate component of proteoglycans. Several types of sulfotransferases determine the pattern of CS sulfation, and thus regulate the biological functions of proteoglycans. The protochordate ascidians are the closest relatives of vertebrates, but the functions of their sulfotransferases have not been investigated. Here, we show that two chondroitin 4‐O‐sulfotransferases (C4STs) play important roles in the embryonic morphogenesis of the ascidian Ciona intestinalis. Ci‐C4ST‐like1 is predominantly expressed in the epidermis and muscle. Epidermal and muscle cells became spherical upon the injection of a Ci‐C4ST‐like1‐specific morpholino oligo (MO), thus suggesting weakened cell adhesion. Co‐injection of a Ci‐C4ST‐like1‐expressing transgene rescued the phenotype, suggesting that the effects of the MO were specific. Ci‐C4ST‐like3 was expressed in the central nervous system, muscle, and mesenchyme. A specific MO appeared to affect cell adhesion in the epidermis and muscle. Convergent extension movement of notochordal cells was also impaired. Forced expression of Ci‐C4ST‐like3 restored normal morphogenesis, suggesting that the effects of the MO were specific. The present study suggests that Ci‐C4ST‐like1 and Ci‐C4ST‐like3 are required for cell adhesion mainly in the epidermis and muscle.     

Roles of Xenopus chemokine ligand CXCLh (XCXCLh) in early embryogenesis

Several chemokine molecules control cell movements during early morphogenesis. However, it is unclear whether chemokine molecules affect cell fate. Here, we identified and characterized the CXC‐type chemokine ligand in Xenopus laevis, Xenopus CXCLh (XCXCLh), during early embryogenesis. XCXCLh is expressed in the dorsal vegetal region at the gastrula stage. Both overexpression and knockdown of XCXCLh in the dorsal region inhibited gastrulation. XCXCLh contributed to the attraction of mesendodermal cells and accelerated the reassembly of scratched culture cells. Also, XCXCLh contributed to early endodermal induction. Overexpression of VegTmRNA or high concentrations of calcium ions induced XCXCLh expression. XCXCLh may play roles in both cell movements and differentiation during early Xenopus embryogenesis.     

Complementary and dynamic type II cadherin expression associated with development of the primate visual system

The middle temporal visual area (MT, also known as V5) is a visual association area that is particularly evolved in the primate brain. The MT receives input from the primary visual area (V1), constitutes part of the dorsal visual pathway, and plays an essential role in processing motion. Connections between the MT and V1 in the primate brain are formed after birth, and are related to the maturation of visual system. However, it remains to be determined what molecular mechanisms control the formation and maturation of the visual system. Cadherins are transmembrane proteins, originally isolated as cell adhesion molecules, which have multiple roles in synapse formation and function. To investigate potential involvement of cadherins in development of the primate visual system, we examined type II cadherin expression (cadherin‐6, ‐8, ‐12) in cortical and thalamic visual areas of pre‐ and postnatal brains of the common marmoset (Callithrix jacchus). In the prenatal brain, cadherin‐6 was dominantly expressed in the pulvino‐MT pathway whereas cadherin‐8 was dominant in the lateral geniculate nucleus (LGN)‐V1 pathway. During postnatal development, there was a downregulation of cadherin‐6 and upregulation of cadherin‐8 expression in the MT. The timing of this cadherin exchange preceded the development of V1‐MT connections. Our results suggest the possibility that changes in cadherin expression are involved in the development of the primate visual system, and that a switch in cadherin expression may be a general mechanism to control neural plasticity of highly cognitive abilities.     

Characterization of sFRP2‐like in amphioxus: insights into the evolutionary conservation of Wnt antagonizing function

Wnt signaling plays a key role in embryonic patterning and morphogenetic movements. The secreted Frizzled‐related proteins (sFRPs) antagonize Wnt signaling, but their roles in development are poorly understood. To determine whether function of sFRPs is conserved between amphioxus and vertebrates, we characterized sFRP2‐like function in the amphioxus, Branchiostoma belcheri tsingtauense (B. belcheri). As in other species of Branchiostome, in B. belcheri, expression of sFRP2‐like is restricted to the mesendoderm during gastrulation and to the anterior mesoderm and endoderm during neurulation. Functional analyses in frog (Xenopus laevis) indicate that amphioxus sFRP2‐like potently inhibits both canonical and non‐canonical Wnts. Thus, sFRP‐2 probably functions in amphioxus embryos to inhibit Wnt signaling anteriorly. Moreover, dorsal overexpression of amphioxus sFRP2‐like in Xenopus embryos, like inhibition of Wnt11, blocks gastrulation movements. This implies that sFRP2‐like may also modulate Wnt signaling during gastrulation movements in amphioxus.     

Paraxial protocadherin mediates cell sorting and tissue morphogenesis by regulating C-cadherin adhesion activity

Little is known about how protocadherins function in cell adhesion and tissue development. Paraxial protocadherin (PAPC) controls cell sorting and morphogenetic movements in the Xenopus laevis embryo. We find that PAPC mediates these functions by down-regulating the adhesion activity of C-cadherin. Expression of exogenous C-cadherin reverses PAPC-induced cell sorting and gastrulation defects. Moreover, loss of endogenous PAPC results in elevated C-cadherin adhesion activity in the dorsal mesoderm and interferes with the normal blastopore closure, a defect that can be rescued by a dominant-negative C-cadherin mutant. Importantly, activin induces PAPC expression, and PAPC is required for activin-induced regulation of C-cadherin adhesion activity and explant morphogenesis. Signaling through Frizzled-7 is not required for PAPC regulation of C-cadherin, suggesting that C-cadherin regulation and Frizzled-7 signaling are two distinct branches of the PAPC pathway that induce morphogenetic movements. Thus, spatial regulation of classical cadherin adhesive function by local expression of a protocadherin is a novel mechanism for controlling cell sorting and tissue morphogenesis.     

H‐Cadherin expression reduces invasion of malignant melanoma

Melanocytic behavior, survival, and proliferation are regulated through a complex system of cell–cell adhesion molecules. Pathologic changes leading to development of malignant melanoma, upset the delicate homeostatic balance between melanocytes and keratinocytes and can lead to altered expression of cell–cell adhesion and cell–cell communication molecules. Malignant transformation of melanocytes frequently coincides with loss of E‐cadherin expression. We now show loss of another member of the superfamily of classical cadherins, H‐cadherin (CDH13), which may be involved in the development of malignant melanoma. The provided data show that H‐cadherin expression is lost in nearly 80% of the analyzed melanoma cell lines. Knockdown of H‐cadherin using siRNA increases invasive capacity in melanocytes. Functional assays show that the re‐expression of H‐cadherin decreases migration and invasion capacity, as well as anchorage‐independent growth in comparison to control melanoma cells. Furthermore, melanoma cells, which re‐express H‐cadherin via stable transfection show a reduction in rate of tumor growth in a nu/nu mouse tumor model in comparison to the parental control transfected cell lines. Our study presents for the first time the down‐regulation of H‐cadherin in malignant melanomas and its possible functional relevance in maintenance healthy skin architecture.     

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.     

The cell adhesion-associated protein Git2 regulates morphogenetic movements during zebrafish embryonic development

Signaling through cell adhesion complexes plays a critical role in coordinating cytoskeletal remodeling necessary for efficient cell migration. During embryonic development, normal morphogenesis depends on a series of concerted cell movements; but the roles of cell adhesion signaling during these movements are poorly understood. The transparent zebrafish embryo provides an excellent system to study cell migration during development. Here, we have identified zebrafish git2a and git2b, two new members of the GIT family of genes that encode ArfGAP proteins associated with cell adhesions. Loss-of-function studies revealed an essential role for Git2a in zebrafish cell movements during gastrulation. Time-lapse microscopy analysis demonstrated that antisense depletion of Git2a greatly reduced or arrested cell migration towards the vegetal pole of the embryo. These defects were rescued by expression of chicken GIT2, indicating a specific and conserved role for Git2 in controlling embryonic cell movements. Git2a knockdown embryos showed defects in cell morphology that were associated with reduced cell contractility. We show that Git2a is required for phosphorylation of myosin light chain (MLC), which regulates myosin II-mediated cell contractility. Consistent with this, embryos treated with Blebbistatin-a small molecule inhibitor for myosin II activity-exhibited cell movement defects similar to git2a knockdown embryos. These observations provide in vivo evidence of a physiologic role for Git2a in regulating cell morphogenesis and directed cell migration via myosin II activation during zebrafish embryonic development.     

RhoA/ROCK and Cdc42 regulate cell‐cell contact and N‐cadherin protein level during neurodetermination of P19 embryonal stem cells

RhoGTPases regulate actin‐based signaling cascades and cellular contacts. In neurogenesis, their action modulates cell migration, neuritogenesis, and synaptogenesis. Murine P19 embryonal stem cells differentiate to neurons upon aggregation in the presence of retinoic acid, and we previously showed that RhoA and Cdc42 RhoGTPases are sequentially up‐regulated during neuroinduction, suggesting a role at this very early developmental stage. In this work, incubation of differentiating P19 cells with C3 toxin resulted in decreased aggregate cohesion and cadherin protein level. In contrast, C3 effects were not observed in cells overexpressing recombinant dominant active RhoA. On the other hand, C3 did not affect cadherin in uninduced cells and their postmitotic neuronal derivatives, respectively expressing E‐ and N‐cadherin. RhoA is thus influential on cell aggregation and cadherin expression during a sensitive time window that corresponds to the switch of E‐ to N‐cadherin. Cell treatment with Y27632 inhibitor of Rho‐associated‐kinase ROCK, or advanced overexpression of Cdc42 by gene transfer of a constitutively active form of the protein reproduced C3 effects. RhoA‐antisense RNA also reduced cadherin level and the size of cell aggregates, and increased the generation of fibroblast‐like cells relative to neurons following neuroinduction. Colchicin, a microtubule disrupter, but not cytochalasin B actin poison, importantly decreased cadherin in neurodifferentiating cells. Overall, our results indicate that the RhoA/ROCK pathway regulates cadherin protein level and cell‐cell interactions during neurodetermination, with an impact on the efficiency of the process. The effect on cadherin seems to involve microtubules. The importance of correct timing of RhoA and Cdc42 functional expression in neurogenesis is also raised. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 289–307, 2004     

Microsporidia Can Acquire Lamin‐like Intermediate Filaments and Cell Adhesion Catenin‐cadherin Complexes from the Host (?)

On their spore surfaces, Microsporidia often develop a canopy of filaments with characteristics of intermediate filaments (IF), as we demonstrated in previous studies on Thelohania sp., Ameson michaelis, and Spraguea lophii. Genomic studies indicate that among invertebrates, lamins that may localize in the cytoplasm or nucleus, are the only known IF type. These IFs can bind to the substrate containing cell adhesion molecules (CAMs) cadherins, associated with β and γ catenins. The objects of this study were to determine whether microsporidia have CAMs with the attached IFs on their envelopes and to find out if these proteins are provided by the host. An examination was made for localization of lamins and CAMs on the spores of the mentioned above species and Anncaliia algerae, plus in the host animals. Then, we determined whether the spores of A. michaelis and A. algerae could bind vertebrate nuclear lamin onto the spore surface. We also tested transgenic Drosophila melanogaster stocks bearing cadherin‐GFP to see whether developing A. algerae parasites in these hosts could acquire host CAMs. The tests were positive for all these experiments. We hypothesize that microsporidia are able to acquire host lamin IFs and cell adhesion catenin–cadherin complexes from the host.     

Expression pattern of E‐cadherin during development of the first tooth in zebrafish (Danio rerio)

Although the importance of cell adhesion in morphogenesis is already known for quite some time, there are remarkably few studies on the distribution and function of adhesion molecules in tooth development. We have chosen the zebrafish to study the role of specific cell adhesion molecules in the development and renewal of teeth. Zebrafish lack an oral dentition but have pharyngeal teeth which are renewed throughout life. Here we focus on the expression of E (epithelial)‐cadherin during the development of the first tooth to develop in the dentition, ‘initiator tooth’ 4V¹. E‐cadherin is expressed exclusively in the pharyngeal epithelium and in the enamel organ throughout all stages of development of this first‐generation tooth. Further studies are needed to compare this expression pattern with protein distribution, both in this and other first‐generation teeth as well as in replacement teeth.     

CHARACTERIZATION OF A RABE (RAS GENE FROM RAT BRAIN E) GTPASE EXPRESSED DURING MORPHOGENESIS IN THE UNICELLULAR GREEN ALGA MICRASTERIAS DENTICULATA (ZYGNEMATOPHYCEAE,STREPTOPHYTA)1

Rab GTPases are central regulators of cell shape in land plants by coordinating vesicle trafficking during morphogenesis. To date, relatively little is known about the role of these ubiquitous signaling proteins during cell growth in microalgae, in particular in the related charophyte algae. This article identifies the first charophyte Rab GTPase, MdRABE1, in Micrasterias denticulata Bréb., a convenient model organism for studying morphogenesis. Its expression correlated with the onset of morphogenesis, and structural analysis indicated that it belongs to the RABE (Ras gene from rat brain E) subclass. Confocal fluorescence and immunoelectron microscopy (IEM) of transiently GFP‐MdRABE1 overexpressing interphase cells demonstrated that the GFP‐MdRABE1 protein was localized to the endoplasmic reticulum, dictyosomes, exocytotic vesicles, the cell margin, the membranes of cell organelles, and in the isthmus zone around the nucleus. Although overexpression phenotyping of both N‐ and C‐terminal green fluorescent protein (GFP) fusions failed to indicate additional functional evidence of the MdRABE1 protein due to mortality of those transgenic cells, its expression profile, bioinformatics, and intracellular localization suggest a role in vesicle trafficking during morphogenesis.     

AGAMOUS‐LIKE13, a putative ancestor for the E functional genes,specifies male and female gametophyte morphogenesis

Arabidopsis AGL13 is a member of the AGL6 clade of the MADS box gene family. GUS activity was specifically detected from the initiation to maturation of both pollen and ovules in AGL13:GUS Arabidopsis. The sterility of the flower with defective pollen and ovules was found in AGL13 RNAi knockdown and AGL13 + SRDX dominant‐negative mutants. These results indicate that AGL13 acts as an activator in regulation of early initiation and further development of pollen and ovules. The production of similar floral organ defects in the severe AGL13 + SRDX and SEP2 + SRDX plants and the similar enhancement of AG nuclear localization efficiency by AGL13 and SEP3 proteins suggest a similar function for AGL13 and E functional SEP proteins. Additional fluorescence resonance energy transfer (FRET) analysis indicated that, similar to SEP proteins, AGL13 is able to interact with AG to form quartet‐like complexes (AGL13–AG)₂ and interact with AG–AP3–PI to form a higher‐order heterotetrameric complex (AGL13–AG–AP3–PI). Through these complexes, AGL13 and AG could regulate the expression of similar downstream genes involved in pollen morphogenesis, anther cell layer formation and the ovule development. AGL13 also regulates AG/AP3/PI expression by positive regulatory feedback loops and suppresses its own expression through negative regulatory feedback loops by activating AGL6, which acts as a repressor of AGL13. Our data suggest that AGL13 is likely a putative ancestor for the E functional genes which specifies male and female gametophyte morphogenesis in plants during evolution.     

Epithelial oral mucosal cells: Do they behave differently when exposed to oral carcinogens?

Objective

To assess the level of maturation and proliferation of epithelial cells and the correlation with immunocytochemical expression of adhesion (E‐cadherin) and cell differentiation (involucrin) markers.

Methods

Cytopathological samples were obtained from four groups of patients: control (CG, n=30); alcohol/tobacco (ATG, n=31), leucoplakia (LG, n=31), and squamous cell carcinoma (SCCG, n=22). Cytopathological smears were collected from all groups for AgNOR, Papanicolaou and immunocytochemical staining.

Results

There was an increase in anucleated cells in ATG compared to CG and in LG compared to lesion‐free groups (P<.05). In addition, there was a higher rate of intermediate cells in lesion‐free groups than in LG (P=.001). When these findings were correlated with positive E‐cadherin expression, there was a smaller number of anucleated and intermediate cells (P<.05). The proliferation rate was higher in the SCCG than in the CG (P<.05) and in the ATG compared to LG (P<.05). Moreover, cell proliferation increased in the presence of positive E‐cadherin expression in the ATG and LG. No statistically significant results were obtained for involucrin analysis.

Conclusion

Cytopathology combined with quantitative techniques such as Papanicolaou, AgNOR, and immunocytochemical expression of E‐cadherin detects changes associated with oral carcinogenesis. The innovative approach used in this study allows assessing the expression of cell adhesion (E‐cadherin) and differentiation (involucrin) markers by means of oral mucosal cytopathology. The E‐cadherin imunocytochemical expression indicated changes associated with the oral carcinogenesis process. An increase in cell proliferation rate in oral squamous cell carcinoma group was associated with the lower immunoexpression of E‐cadherin. Cytopathology combined with quantitative techniques and immunocytochemical expression of E‐cadherin may detect early alterations associated with oral carcinogenesis.     

Contribution of Cadherins to Directional Cell Migration and Histogenesis in Xenopus Embryos

Perturbation of adhesion mediated by cadherins was achieved by over-expressing truncated forms of E- and EP-cadherins (in which the extracellular domain was deleted) in different blastomeres of stage 6 Xenopus laevis embryos. Injections of mRNA encoding truncated E- and EP-cadherins into A1A2 blastomeres resulted in inhibition of cell adhesion and, at later stages, in morphogenetic defects in the anterior neural tissues to which they mainly contribute. In addition, truncated EP-cadherin mRNA produced a duplication of the dorso-posterior axis in a significant number of cases. The expression of truncated E- and EP-cadherins in blastomeres involved in gastrulation and neural induction (B1B2 and C1), led to the duplication of the dorso-posterior axis as well as to defects in anterior structures. Morphogenetic defects obtained with truncated EP-cadherin were more severe than those induced with truncated E-cadherin. Cells derived from blastomeres injected with truncated EP-cadherin mRNA, dispersed more readily at the blastula and gastrula stages than the cells derived from the blastomeres expressing truncated E-cadherin. Presumptive mesodermal cells expressing truncated cadherins did not engage in coherent directional migration. The alteration of cadherin-mediated cell adhesion led directly to the perturbation of the convergent-extension movements during gastrulation as shown in the animal cap assays and indirectly to perturbation of neural induction. Although the cytoplasmic domains of type I cadherins share a high degree of sequence identity, the over-expression of their cytoplasmic domains induces a distinct pattern of perturbations, strongly suggesting that in vivo, each cadherin may transduce a specific adhesive signal. These graded perturbations may in part result from the relative ability of each cadherin cytoplasmic domain to titer the P-catenin.